Intermediate steps in the formation of neuronal SNARE complexes

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.


Figure S1
. Syntaxin and SNAP25 form a 2:1 complex.(a) Elution profiles of 5 µM of syntaxin (red curve, upper graph), SNAP25 (green curve, middle graph) and the syntaxin-SNAP25 complex (black curve, bottom graph).The samples were primarily incubated overnight at 4°C, which was followed by size exclusion chromatography at the same temperature.The elution profile of syntaxin shows oligomer formation with three distinguishable peaks at approximately 11 ml, 13.2 ml and 14.5 ml, that presumably correspond to syntaxin tetramer, dimer and monomer, respectively.The elution profile of the syntaxin-SNAP25 sample contains free SNAP25 (unmarked peak in the bottom graph), but no free syntaxin, consistent with the formation of the 2:1 complex.The relevant peaks (indicated with symbols) were analyzed by Coomassie stained SDS-PAGE and are shown next to the corresponding chromatogram.The original image was converted to a greyscale image.Both SDS-PAGE images originate from the same gel and contain the same gel ladder, with the bottom image being cropped as indicated by the dotted line.The top SDS-PAGE image shows the ladder and lines 1-3, while the bottom SDS-PAGE image shows again the ladder and line 11 of the same gel.(b) Equimolar titration traces of syntaxin and SNAP25 were fit to a monoexponential equation:  =  (−  ) + ; where  corresponds to the fluorescence intensity,  to the fluorescence amplitude,  to the time,  to the starting point of the fit, and   to the observed rate constant.(c, d, e) Plots of the square of the apparent rate constant (kobs 2 ) and the initial concentration of syntaxin ([syntaxin]0) for every experiment can be best fit with a hyperbolic (dashed curves), rather than a linear function (full line), indicating that syntaxin and SNAP25 binding is not in 1:1 stoichiometry and thus does not occur in a single step reaction.Fitting was performed using the Generalized Hyperbola function (OriginPro 2019b, OriginLab Corporation) ; where y corresponds to fluorescence intensity, x to the initial syntaxin concentration, while a, b, c, and d are the parameters determining the shape of the hyperbola: a corresponds to the horizontal asymptote; for d>0 hyperbola has a vertical asymptote equal to 1  , for d<0 hyperbola has an oblique asymptote with the slope of ( × ) and an intercept of ( − ).

Figure S4. Addition of a 10-fold excess of unlabeled syntaxin or SNAP25 to labeled syntaxin and SNAP25 at equilibrium yield opposite FRET changes that are due to the low starting concentration of the 2:1 complex. (a) Donor-labeled syntaxin (green dots) and acceptor-labeled SNAP25 (red dots)
were incubated at equimolar concentrations to form a 2:1 complex.After equilibration (approx.10 min), unlabeled SNAP25 was added and the change in the acceptor fluorescence was monitored over time.Final concentrations were 1 µM for the labeled proteins and 10 µM for unlabeled SNAP25.(b) Addition of unlabeled SNAP25 caused a decrease in the acceptor fluorescence as the labeled SNAP25 in the 2:1 complex is replaced with unlabeled SNAP25 (green trace).The slight decrease observed in the buffer control is caused by dissociation due to dilution of the complex.(c) Mono-exponential fit (see above) of the dissociation yielded a koff of (3.44 ± 0,02) x 10 -3 s -1 .(e) Same as in a) but this time excess unlabeled syntaxin (7.5 μM final concentration) was added to donor-labeled syntaxin (0.75 μM, green dots) and acceptor-labeled SNAP25 (0.75 μM, red dots).(f) In contrast to b), an increase rather than a decrease of the acceptor fluorescence (red trace) was observed that lasted ~600 s, and was absent from controls, indicating that for saturating SNAP25 in the 2:1 complex much higher concentrations of syntaxin are required.

Figure S10. Analysis of SNARE sub-complexes using size-exclusion chromatography following an overnight incubation of SNARE mixtures in comparison to free SNAREs. (a, c) Syntaxin and SN1
alone and with synaptobrevin show formation of a stable complex as indicated by sharp peaks in the chromatograms eluting at ~11.6 ml (marked with a diamond) and ~8.8 ml (marked with a star), respectively.(b, d) While there was no indication of a complex formation between syntaxin and SN2, addition of synaptobrevin yielded two peaks in the chromatogram at ~9.5 ml (marked with a star) and ~11.2 ml (marked with a circle), consistent with heterogeneous and unstable complex that likely dissociates during the size exclusion run.(green) and presence of syntaxin (1-250) (red).Each of the six single-labeled SN2 fragments are at a final concentration of 24 M and syntaxin is at 32 M.(c) Normalized X-band EPR spectra of 24 M SNAP25 SN1 (7-83) labels in the absence (green) and presence (black) of 24 M SN2 (141-204) (d) Normalized X-band EPR spectra of labeled synaptobrevin in various conditions.25 M isolated synaptobrevin is shown in blue.25 M synaptobrevin in the presence of 28 M syntaxin is in red.25 M synaptobrevin in the presence of 28 M SN1 is in green.25 M synaptobrevin in the presence of 28 M SN1 and 28 M syntaxin is in gray.28R1 and 93R1 synaptobrevin, and the added proteins follow the concentration scheme described above.55R1 synaptobrevin is at 26 M in all four conditions.When SN1 and syntaxin are added to 55R1 synaptobrevin, both proteins are at 30 M.(e) Normalized X-band EPR spectra of labeled synaptobrevin in various conditions.25 M isolated synaptobrevin is shown in blue.25 M synaptobrevin in the presence of 28 M syntaxin is in red.25 M synaptobrevin in the presence of 28 M SN2 is in green.25 M synaptobrevin in the presence of 28 M SN2 and 28 M syntaxin is in black.28R1 and 93R1 synaptobrevin, and the added proteins follow the concentration scheme described above.55R1 synaptobrevin is at 26 M in all four conditions.When SN2 and syntaxin are added to 55R1 synaptobrevin, both proteins are at 30 M.

Figure S2 .
Figure S2.Low syntaxin dimer concentration leads to lower fluorescence amplitudes and therefore lower 2:1 complex formation.(a) Monoexponential fit of the traces from SNAP25 titration over syntaxin experiment and (b) syntaxin titration over SNAP25 experiment.The traces were fit to the following equation:  =  (−  ) +  (see above).Comparison between syntaxin and SNAP25 titrations show similar noise level at the lowest (c) and highest (d) titrant concentrations.In the case of SNAP25 titration (green traces), the low concentrations of syntaxin dimer causes the reaction to reach only a fraction of the amplitude achieved with syntaxin titration (red traces).

Figure S6 .
Figure S6.Synaptobrevin does not form complexes with syntaxin, or SNAP25.(a) Schematic representation of equimolar titrations of donor-labeled syntaxin and acceptor-labeled synaptobrevin, and (c) donor-labeled SNAP25 and acceptor-labeled synaptobrevin.Upon triggering, equal volumes of the labeled proteins was mixed and the interaction was monitored in the recording cell.Titration traces of both experiments show no difference to the control even at the highest concentrations of 1.5 µM, showing that synaptobrevin does not interact with either SNAP25 or syntaxin alone.(b, d) Size exclusion chromatography of syntaxin-synaptobrevin, and SNAP25-synaptobrevin (5 μM each), respectively, after incubation overnight at 4°C, showed only superimposition of the free monomer elution profiles, confirming the absence of heterooligomeric complexes.

Figure S7 .
Figure S7.SNARE complex formation in the presence of excess SNAP25 can be fit to a monoexponential function.(a) Excess of unlabeled SNAP25 was added to premixed donor-labeled syntaxin and acceptor-labeled synaptobrevin (b) titration traces showed an increase that corresponded to the increase in the concentration of SNAP25, while all the controls remained flat.(c) The traces were best fit to a monoexponential equation ( =  (−  ) + , see above) and (d) the obtained kobs showed a linear increase with increasing SNAP25 concentrations.The three different shades of green (d) correspond to three repetitions of the experiment.

Figure S8 .
Figure S8.SNARE complex formation in the presence of excess of syntaxin allows for capturing of two kinetically distinct phases.(a) Excess of unlabeled syntaxin was titrated over pre-mixed donorlabeled SNAP25 and acceptor-labeled synaptobrevin.(b) The titration traces show a robust increase that was absent from all controls.(c) Two phases could be distinguished in the titration traces that were then fit to a biexponential equation ( =  1  (−  1 ) +  2  (−  2 ) + , see above), yielding k1obs and k2obs.(d, e) Both detected phases showed linear dependence of the initial syntaxin concentration with the first phase (described by k1obs) being compatible with binding of SNAP25 to syntaxin dimer as shown in main text in Figure 2c, and the second phase compatible to the SNARE complex formation.The three different colors (d, e) correspond to three repetitions of the experiment.

Figure S11 .
Figure S11.Continuous Wave Electron Paramagnetic Resonance Analysis.(a) Normalized X-band EPR spectra of single SNAP25 SN1 (7-83) labels in the absence (green) and presence of syntaxin (1-250) (red).Each of the six single-labeled SN1 fragments are at a final concentration of 24 M and syntaxin is at 32 M.(b) Normalized X-band EPR spectra of single SNAP25 SN2 labels in the absence